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Application of soybean protein kinase gene GmSTK_IRAK

A soybean protein and gene technology, applied in the fields of application, genetic engineering, enzymes, etc., can solve problems that have not been reported yet, and achieve the effect of good application prospects

Active Publication Date: 2021-02-19
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the study of GmSTK_IRAK gene in soybean has not been reported so far

Method used

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  • Application of soybean protein kinase gene GmSTK_IRAK
  • Application of soybean protein kinase gene GmSTK_IRAK
  • Application of soybean protein kinase gene GmSTK_IRAK

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Cloning of Soybean GmSTK_IRAK Gene and Construction of Plant Expression Vector

[0035] (1) Design primers, extract RNA, and reverse transcribe cDNA:

[0036] The total RNA of soybean Williams 82 leaves was extracted with a plant total RNA extraction kit (DP432, Tiangen), and the integrity of the RNA was detected by 1% agarose gel electrophoresis.

[0037] The cDNA was synthesized according to the instructions of the TaKaRa Primer Script TMRT reagent kit with gDNA Eraser kit.

[0038] (2) PCR amplification:

[0039] Step 1: Prepare PCR reaction solution (50μl system) according to the following components: 10×PCR Buffer (25μl), ddH 2 O (9 μl), dNTP (10 μl), GmSTK_IRAK-F (1.5 μl), GmSTK_IRAK-R (1.5 μl), cDNA (2 μl), KODFX enzyme (1 μl);

[0040] The designed primers are:

[0041] GmSTK_IRAK-F:

[0042] 5'-CCTACCACATTAATTACTCACTCTTCACTCA-3'

[0043] GmSTK_IRAK-R:

[0044] 5'-TCAACTTTAACGCTCATTCCTGCATTCAT-3'

[0045] Step 2: The reaction was carried out on a BIO-RAD...

Embodiment 2

[0059] Cultivation of GmSTK_IRAK Gene Overexpression and Silencing Transgenic Soybean

[0060] (1) Disinfection and germination of seeds

[0061] The surface disinfection of soybean seeds was sterilized by dry chlorine gas. Pick clean seeds that are mature and plump, without disease spots, and without hardness, and arrange them in a single layer in a 90×15mm petri dish. Open the petri dish and put it in a desiccator. Place a 500ml glass beaker in the desiccator. Measure 75ml of commercial bleach with a 100ml graduated cylinder and add it to the beaker. Measure 3ml of 12M HCl with a 10ml graduated cylinder and slowly add it along the wall of the beaker. Cover the lid of the desiccator to ensure that the container is sealed, and let it stand overnight for 10-16 hours. After the sterilization is completed, transfer the lid of the culture dish to a sterile ultra-clean table, open the lid of the culture dish, and blow it with strong wind for 25-40 minutes to remove it. Chlorine g...

Embodiment 3

[0074] Verification of genetically modified materials

[0075] Since the vector used for overexpressing the transgene contains the gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), this enzyme can block the interference of glyphosate on the biosynthetic pathway, so that it will not be killed by glyphosate . The herbicide glyphosate used for identification was diluted 1000 times (concentration: 200mg / L) and the transgenic seedlings were sprayed. The negative plants withered and died, and the positive plants showed obvious resistance and maintained good growth.

[0076] DNA was extracted from the leaves of positive plants that survived the herbicide detection (CTAB Plant Genomic DNA Rapid Extraction Kit: Zhongding Company, Cat. No. DN14-100T), and positive materials were further screened by PCR detection of the marker EPSPS gene. The EPSPS gene primer sequence is:

[0077] Upstream primer: 5'-AGGACGTCATCAATACGGGC-3'

[0078] Downstream primer: 5'-ATCCACGCCATT...

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Abstract

The invention provides application of a soybean protein kinase gene GmSTK_IRAK, and belongs to the technical field of plant genetic engineering. A soybean GmSTK_IRAK gene is cloned through PCR, through transgenosis and a gene editing technology, an over-expression GmSTK_IRAK and GmSTK_IRAK gene silencing transgenosis plant is obtained, the phosphorus uptake and utilization efficiency, the biomassand the yield of the GmSTK_IRAK over-expression transgenosis soybean are improved by a large margin, and the phosphorus uptake and utilization efficiency, the biomass and the yield of a gene silencingsoybean plant are lowered. The soybean protein kinase gene GmSTK_IRAK disclosed by the invention can be delivered into the plant as a target gene, phosphorus metabolism and balance capacity in a transgenosis plant body is regulated, and the soybean protein kinase gene GmSTK_IRAK has an important meaning for culturing high phosphorus efficiency soybean new species.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, in particular to the application of a soybean protein kinase gene GmSTK_IRAK. Background technique [0002] Soybean is a crop that requires a large amount of phosphorus, and its grain phosphorus content is much higher than that of rice, wheat, and corn (Li Xihuan et al., 2011). Phosphorus deficiency not only affects the growth and development of soybean, but also significantly affects the formation of root nodules, and eventually Reduce yield (Wang Shuqi et al., 2010). Soil phosphorus deficiency has become an important factor limiting soybean production in my country (Liu Haixu et al., 2017). In order to solve the contradiction between the high phosphorus demand of soybean and the low soil available phosphorus, isolate and identify new key genes for low phosphorus tolerance, use genetic engineering to introduce low phosphorus sensitive varieties, and carry out phosphorus effici...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/54
CPCC12N15/8261C12N15/8218C12N15/8216C12N9/1205C12Y207/11001C12N9/12C12N2310/20C12N15/8213C12N9/22C12N15/8265
Inventor 张丹杨宇明张恒友吕海燕褚姗姗
Owner HENAN AGRICULTURAL UNIVERSITY
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